Despite intense investigation, detailed molecular events occurring during transmembrane signalling by the insulin receptor remain poorly understood. We have been especially interested in understanding how insulin binds to the receptor and how the binding signal is transmitted to intracellular signalling pathways. However, detailed structural analyses of the insulin receptor and its complex with insulin have not been possible using such methods as x-ray crystallography. In fact, Dodson & colleagues recently demonstrated that crystal structures of insulin depict inactive conformers. Our own studies of insulin structure and dynamics in solution suggest that a significant change in structure accompanies receptor binding (Nature, in press), and that a protein surface not present in insulin crystals is exposed during binding and buried again within the insulin-receptor complex. Experiments are proposed to test this hypothesis and determine directly which residues of insulin and the insulin receptor interact at this important protein-- protein interface, including: (1) Multi-dimensional, isotope-assisted NMR studies of selected analogues which we hypothesized exist in configurations approximating the structure of insulin at the receptor surface. (2) Residues of insulin that are thought to be buried in the complex will be substituted with benzoylphenylalanine (Bpa), a photoactivatable amino acid, for direct confirmation of insulin positions that are in contact with the receptor. (3) Bpa-insulin analogues cross-link the insulin receptor with unusually high efficiency (70-90%); cross-linked receptors will be fragmented and sequenced to determine receptor residues that are actually buried within the complex. (4) Identified residues will be mutated to confirm effects on binding and test effects on signalling; use of tryptophan for substitutions will facilitate fluorescence energy transfer experiments with dansyl-insulin analogues to further confirm proximity to the binding pocket. Results from these studies will be used to model a binding interface between insulin and the insulin receptor. These findings will dramatically improve our understanding of structural relationships between insulin and the insulin receptor and mechanisms of insulin-stimulated receptor signalling, which is of fundamental importance for understanding normal and abnormal receptor functioning in health and disease.